We have demonstrated that hot water extracts of sclerotia of Sclerotinia sclerotiorum IFO 9395 (TSHW) show various immunomodulating activities and mitogenic substance(s) were recovered from the beta-1,3-glucanase resistant-fraction (EDP) (Shinohara et al. Chem. Pharm. Bull., 37, 2174 (1989]. In this paper, we examined whether or not the mitogenic substance(s) were also obtained from the other methods, phosphate buffer extraction. Although the native extracts (3S-M) sterilized with a membrane filter showed a slight mitogenicity to murine splenocytes, 3S-M denatured in boiling water (3S-MB) showed significant activity. Treatment of 3S-M for only 1 min in boiling water or 10 min at 70 degrees C was sufficient to show significant mitogenic activity. After heat treatment of 3S-M in boiling water for 30 s, the main band corresponding to that of 3S-M was not clearly observed. Instead, new bands appeared at the top of the gel in normal-polyacrylamide gel electrophoresis (normal-PAGE), suggesting that many physicochemical changes occurred during the heat treatment. These findings suggest that heat denaturation of the substance(s) from sclerotia was one of the triggering mechanisms expressing mitogenic activity to murine splenocytes.